羊毛脂固醇對 α-crystallin 和 SM 細胞膜間作用的影響

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張家綾(Chia-Ling Chang) 查詢紙本館藏

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物理學系

論文名稱

羊毛脂固醇對 α-crystallin 和 SM 細胞膜間作用的影響
(Influence of lanosterol on the interaction between α-crystallin and SM membranes)

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★ 細胞膜媒介麥角固醇與 α-crystallin 的作用

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摘要(中)

白內障是老年人的常見疾病，通常會伴隨著失明的風險，白內障是眼睛水晶體中蛋白質異常聚集造成混濁，使進入水晶體的光線發生散射所造成。人類水晶體中最豐富的蛋白質是由 α-、β- 和 γ-晶狀蛋白所組成的，先前的研究表明 α-晶狀蛋白具有伴護分子的功能，可以防止 β- 和 γ-晶狀蛋白錯誤折疊導致蛋白質變性而聚集。研究顯示，α-晶狀蛋白與水晶體脂質膜的結合能力隨著年齡的增長而增加，並且觀察到 α-晶狀蛋白與錯誤折疊或展開的蛋白質和細胞膜之間的關聯性高。此外，最近的研究表明，固醇不僅可以保持水晶體透明度，還可以恢復混濁的水晶體。由於固醇和脂質膜之間有很強的相互作用，因此我們提出一個由細胞膜媒介固醇和 α-晶狀蛋白作用的模型來說明此機制。
α-晶狀蛋白主要由 αA- 和 αB-晶狀蛋白兩個亞基所構成。在本論文中，我們使用基因轉殖技術來表現蛋白質，然後進行蛋白質純化來得到 αA- 和 αB-晶狀蛋白，並使用從雞蛋中提取的 SM(sphingomyelin) 脂質和羊毛脂固醇，作為細胞膜模型系統來研究 α-晶狀蛋白、細胞膜和固醇間的相互作用。利用溶菌酶(lysozyme)和乙醇脫氫酶(alcohol dehydrogenase, ADH)兩種標準蛋白作為標準，測量蛋白質的聚集程度來決定 α-晶狀蛋白的抗聚集能力。通過圓二色光譜儀(circular dichroism, CD)測定 α-晶狀蛋白與細胞膜結合後二級結構的變化，以測量水溶液中與細胞膜結合 α-晶狀蛋白的比例。利用小角 X 射線散射(small angle x-ray scattering, SAXS)測量細胞膜與 α-晶狀蛋白結合後細胞膜結構變化。
根據實驗結果的結構和功能數據，本研究將討論羊毛脂固醇對 α-晶狀蛋白和 SM 脂質膜之間相互作用的影響。結果表明 SM 脂質膜會抑制 α-晶狀蛋白的抗聚集能力，當在細胞膜中添加羊毛脂固醇時，不僅使 SM 脂質膜增厚，還可以恢復 α-晶狀蛋白的抗聚集能力。

摘要(英)

Cataract, a common disease for elder people, usually accompanies the risk of blindness. It is induced by light scattering from the clouding of the abnormal aggregation of proteins in the eye lens. The most abundant proteins in the human lens are crystallin composed of α-, β- and γ-crystallins. The previous studies have indicated α-crystallin exhibits chaperone-like activity to prevent β- and γ-crystallins from misfolding induced aggregation.
The previous studies have reported that the binding capacity of α-crystallins to lipids membranes in the lens increases with age. Moreover, recent evidence showed that sterols can not only maintain lens transparency but also recover lens clouding. Due to the strong interaction between sterols and lipid membranes, we proposed a model based on the membrane-mediated interaction between sterols and α-crystallins to illustrate the mechanism.
The α-crystallins are mainly composed of two relative subunits, αA- and αB-crystallin. In this study, we used gene transcription to express protein and produced αA- and αB-crystallin proteins by protein purification. SM lipids extracted from egg and lanosterol were used as model systems to investigate the interactions between α-crystallins, membranes, and sterols. Lysozyme and alcohol dehydrogenase (ADH) proteins were used as standard assays to probe the chaperone-like activity. The binding ratio of α-crystallins to membranes was determined via the change of secondary structure probed by circular dichroism (CD). Small-angle X-ray scattering (SAXS) was used to monitor the structural changes of membranes induced by bound α-crystallins.
According to the structural as well as functional data, the effects of lanosterol on the interaction between α-crystallins and SM lipid membranes will be discussed in this study. The result indicates that SM lipid membranes are able to inhibit the anti-aggregation ability of α -crystallins. With adding lanosterol into membranes, not only thickening of SM lipid membranes but also recovering of anti-aggregation ability of α-crystallins was observed.

關鍵字(中)

★ 羊毛脂固醇
★ α-晶狀蛋白
★ 鞘磷脂

關鍵字(英)

★ lanosterol
★ α-crystallin
★ sphingomyelin

論文目次

中文摘要 i
英文摘要 ii
致謝 iii
目錄 iv
圖目錄 vi
表目錄 ix
符號說明 x
第一章緒論 01
1.1 研究背景 01
1.2 水晶體構造及組成 02
1.2.1 水晶體構造 02
1.2.2 晶狀蛋白 03
1.2.3 水晶體細胞膜 05
1.2.4 磷脂質 06
1.2.5 固醇 08
1.3 白內障的形成及類型 09
1.4 研究動機 10
1.5 研究方法 11
第二章材料、樣品製備與實驗方法 13
2.1 材料 13
2.1.1 磷脂質 13
2.1.2 固醇 13
2.1.3 蛋白質 14
2.1.4 基因片段 14
2.1.5 菌 14
2.1.6 藥品 14
2.1.7 儀器設備 15
2.2 實驗樣品製備及其基本原理 18
2.2.1 αA-Crystallin、αB-Crystallin 18
2.2.2 小型單層膜微胞(Small Unilamellar Vesicles, SUVs) 24
2.3 實驗方法及原理 27
2.3.1 粒徑分析儀(Dynamic Light Scattering, DLS) 27
2.3.2 圓二色光譜儀(Circular Dichroism Spectropolarimeter, J-810) 30
2.3.3 蛋白質抗聚集能力(chaperone like activity)測量 38
2.3.4 小角度 X 光散射(Small-angle X-ray scattering, SAXS) 42
第三章實驗結果 51
3.1 α-crystallin 純化 51
3.2 粒徑分析儀 52
3.3 圓二色光譜儀 52
3.3.1 數據分析 52
3.3.2 結果 56
3.4 伴護活性測量結果 58
3.4.1 數據分析 58
3.5 小角度 X 光散射實驗結果 68
3.5.1 數據分析 68
3.5.2 結果 76
第四章結果與討論 80
4.1 α-crystallin 與細胞膜樣品 80
4.2 α-crystallin 與 Egg SM 細胞膜的作用 80
4.3 加入 Lanosterol 的影響 83
第五章結論 85
參考文獻 86

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指導教授

李明道(Ming-Tao Lee)

審核日期

2023-7-25

推文